beta3- adrenergic receptor agonist crystal forms, processes for the production thereof, and uses thereof

ABSTRACT

The present invention provides an anhydrous crystal form of the tosylate salt of (R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol, and a crystal form of the monohydrate of such tosylate salt; processes useful in the preparation of such crystal forms; pharmaceutical compositions comprising such crystal forms; methods of treating β 3 -adrenergic receptor-mediated diseases, conditions, and disorders in a mammal using such crystal forms, or such pharmaceutical compositions; and methods of increasing the content of lean meat in edible animals using such crystal forms, or such pharmaceutical compositions.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/360,252 filed on Feb. 27, 2002 and incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to crystal forms of the anhydroustosylate salt, and the monohydrate of such tosylate salt, of theβ₃-adrenergic receptor agonist(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol;processes for the production of such crystal forms; pharmaceuticalcompositions comprising such crystal forms; and methods of treating,inter alia, diabetes and hypoglycemia, with such crystal forms, or suchpharmaceutical compositions.

BACKGROUND OF THE INVENTION

[0003] Diabetes mellitus is characterized by metabolic defects in theproduction and utilization of carbohydrates which result in the failureto maintain appropriate blood sugar levels. The results of these defectsinclude, inter alia, elevated blood glucose or hyperglycemia. Researchin the treatment of diabetes has centered on attempts to normalizefasting and postprandial blood glucose levels. Current treatmentsinclude administration of exogenous insulin, oral administration ofdrugs, and dietary therapies.

[0004] Two major forms of diabetes mellitus are recognized. Type 1diabetes, or insulin-dependent diabetes mellitus (IDDM), is the resultof an absolute deficiency of insulin, the hormone that regulatescarbohydrate utilization. Type 2 diabetes, or non-insulin-dependentdiabetes mellitus (NIDDM), often occurs with normal, or even elevated,levels of insulin and appears to be the result of the inability oftissues to respond appropriately to insulin. Most Type 2 diabeticpatients are also obese.

[0005] The crystal forms, and the pharmaceutical compositions comprisingsuch crystal forms, of the present invention effectively lower bloodglucose levels when administered orally to mammals with hyperglycemia ordiabetes.

[0006] Obesity constitutes a major health risk that leads to mortalityand incidence of Type 2 diabetes mellitus, hypertension, anddyslipidemia. In the United States, more than 50% of the adultpopulation is overweight, and almost 25% of the population is consideredto be obese. The incidence of obesity is increasing in the United Statesat a three-percent cumulative annual growth rate. While the vastmajority of obesity occurs in the United States and Europe, theprevalence of obesity is also increasing in Japan. Furthermore, obesityis a devastating disease which can also wreak havoc on an individual'smental health and self-esteem, which can ultimately affect a person'sability to interact socially with others. Unfortunately, the preciseetiology of obesity is complex and poorly understood, and societalstereotypes and presumptions regarding obesity only tend to exacerbatethe psychological effects of the disease. Because of the impact ofobesity on society in general, much effort has been expended in effortsto treat obesity, however, success in the long-term treatment and/orprevention thereof remains elusive.

[0007] The crystal forms, and the pharmaceutical compositions comprisingsuch crystal forms, of the present invention also reduce body weight ordecrease weight gain when administered to a mammal. The ability of suchcrystal forms, and such compositions to affect weight gain is due toactivation of β₃-adrenergic receptors which stimulate the metabolism ofadipose tissue.

[0008] β-Adrenergic agents have been generally classified into β₁, β₂,and β₃ receptor-specific subtypes. Agonists of β-receptors generallypromote the activation of adenyl cyclase. Activation of β₁ receptorsinvolves an increase in heart rate while activation of β₂ receptorsinduces smooth muscle tissue relaxation which produces a drop in bloodpressure and the onset of skeletal muscle tremors. Activation of β₃receptors is known to stimulate lipolysis (e.g.; the breakdown ofadipose tissue triglycerides into glycerol and fatty acids) andmetabolic rate (energy expenditure), thereby promoting the loss of fatmass. Accordingly, compounds that stimulate β₃ receptors are useful asanti-obesity agents, and can be further used to increase the content oflean meat in edible animals. In addition, compounds that are β₃ receptoragonists have hypoglycemic activity, however, the precise mechanism ofthis effect is presently unknown.

[0009] Until recently, β₃-adrenergic receptors were thought to be foundpredominantly in adipose tissue, however, β₃ receptors are now known tobe located in such diverse tissues as the intestine (J. Clin. Invest.,91, 344 (1993)), and the brain (Eur. J. Pharm., 219, 193 (1992)).Stimulation of β₃ receptors has also been demonstrated to inducerelaxation of smooth muscle in the colon, trachea, and bronchi. See, forexample, Life Sciences, 44, 1411 (1989), Br. J. Pharm., 112, 55 (1994),and Br. J. Pharmacol., 110, 1311 (1993). Furthermore, stimulation of β₃receptors has also been found to induce relaxation ofhistamine-contracted guinea pig ileum. See, for example, J. Pharm. Exp.Ther., 260, 1, 192 (1992).

[0010] The β₃ receptor is also expressed in the human prostate (J. Clin.Invest., 91, 344 (1993)). Because stimulation of the β₃ receptor causesrelaxation of smooth muscles that have been shown to express the β₃receptor, i.e., intestinal smooth muscle, one of ordinary skill in theart would also predict relaxation of prostate smooth muscle. Therefore,β₃ agonists are useful in the treatment or prevention of prostatedisease.

[0011] Commonly assigned U.S. Pat. No. 5,977,124 discloses certainβ₃-adrenergic receptor agonists having utility in the treatment of,inter alia, hypoglycemia and obesity.

[0012] U.S. Pat. No. 5,776,983 discloses certain catecholamines usefulas β₃ agonists.

[0013] U.S. Pat. No. 5,030,640 discloses certain α-heterocylicethanolamino alkyl indoles, which are useful as growth promoters,bronchodilators, anti-depressants, and anti-obesity agents.

[0014] U.S. Pat. No. 5,019,578 discloses certain α-heterocyclicethanolamines useful as growth promoters.

[0015] U.S. Pat. No. 4,478,849 discloses pharmaceutical compositionscomprising certain ethanolamine derivatives and methods of using suchcompositions in the treatment of obesity and/or hyperglycemia.

[0016] U.S. Pat. No. 4,358,455 discloses certain heterocyclic compoundsof the structural formula Het—CHOH—CH₂—NH-aralkyl, which compounds asuseful for treating glaucoma and cardiovascular disease.

[0017] European Patent Application Publication No. 0 516 349, publishedDec. 2, 1992, discloses certain 2-hydroxyphenethyl amines which possessanti-obesity, hypoglycemic, and related utilities.

[0018] U.S. Pat. No. 5,153,210 discloses certain heterocyclic compoundsof the formula R⁰—X—CH(OH)—CH₂N(R¹)—C(R²)(R³)—(CH₂)_(n)—Y—A—R⁴—R⁵ whichcompounds are useful as anti-obesity and anti-hyperglycaemic agents.

[0019] PCT International Patent Application Publication No. WO 99/65877,published Dec. 23, 1999, discloses heterocyclic compounds having thestructural formula

[0020] which compounds are useful for the treatment of diseasesusceptible to amelioration by the administration of an atypicalβ-adrenoceptor agonist.

[0021] Commonly assigned U.S. Provisional Application No. 60/242,274,filed Oct. 20, 2000, and incorporated herein in its entirety byreference, discloses certain β₃- adrenergic receptor agonists ofstructural Formula (I),

[0022] (I)

[0023] the stereoisomers and prodrugs thereof, and the pharmaceuticallyacceptable salts of the compounds, stereoisomers, and prodrugs,including the aforementioned(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol.

[0024] The present invention provides an anhydrous crystal form of thetosylate salt of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol; acrystal form of the monohydrate of such tosylate salt; processes usefulin the preparation of such crystal forms; pharmaceutical compositionscomprising such crystals forms; methods of treating β₃-adrenergicreceptor-mediated diseases, conditions, and disorders in a mammal usingsuch crystal forms, or such pharmaceutical compositions; and methods ofincreasing lean meat content in an edible animal using such crystalforms, or such pharmaceutical compositions.

SUMMARY OF THE INVENTION

[0025] The present invention provides an anhydrous crystal form of thetosylate salt of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol, anda crystal form of the monohydrate of such tosylate salt; processesuseful in the preparation of such crystal forms; pharmaceuticalcompositions comprising such crystal forms; methods of treatingβ₃-adrenergic receptor-mediated diseases, conditions, and disorders in amammal using such crystal forms, or such pharmaceutical compositions;and methods of increasing the content of lean meat in edible animalsusing such crystal forms, or such pharmaceutical compositions.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention provides an anhydrous crystal form of thetosylate salt of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol, acrystal form of the monohydrate of such salt; processes useful in thepreparation of such crystal forms; pharmaceutical compositionscomprising such crystal forms; methods of treating β₃-adrenergicreceptor-mediated diseases, conditions, and disorders in a mammal usingsuch crystal forms, or such pharmaceutical compositions; and methods ofincreasing the content of lean meat in edible animals using such crystalforms, or such pharmaceutical compositions. Such crystal forms, and suchpharmaceutical compositions, further possess utility for increasing thecontent of lean meat in edible animals, i.e., ungulate animals such ascattle, swine, and the like, as well as poultry.

[0027] As employed throughout the instant description, the phrase“crystal forms of the instant invention” means, as appropriate, acrystal form of the anhydrous tosylate salt and/or a crystal form of themonohydrate of such tosylate salt, of the compound(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol.

[0028] As employed throughout the instant description and appendantclaims, the phrase “therapeutically effective amount” means an amount ofthe crystal forms of the instant invention, or a pharmaceuticalcomposition comprising such crystal forms, which attenuates,ameliorates, prevents, or delays the onset of one or more diseases,conditions, or disorders, and/or one or more symptoms of such diseases,conditions, or disorders.

[0029] The term “mammal” means animals including, for example, dogs,cats cows, sheep, horses, and humans. Preferred mammals include humans,including members of both male and female sexes.

[0030] The phrase “pharmaceutically acceptable” indicates that thesubstance or composition must be compatible chemically and/ortoxicologically with the other ingredients comprising a pharmaceuticalformulation, and/or the mammal, or other animal being treated therewith.

[0031] The terms “treat”, “treating”, or “treatment” embrace bothpreventative, i.e., prophyactic, and palliative treatment.

[0032] The compound(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol maybe prepared as disclosed in the aforementioned U.S. ProvisionalApplication Serial No. 60/242,274. Alternatively,(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol mayalso be prepared according to an exemplary process for preparing acompound of the structural formula

[0033] a pharmaceutically acceptable salt thereof, or a hydrate of thepharmaceutically acceptable salt, which process comprises the steps of:

[0034] (a) reducing an α-bromoketone derivative of the structuralformula

[0035] or an acid addition salt thereof, to form an (R)-bromoalcoholderivative of the structural formula

[0036] (b) protecting the (R)-bromoalcohol derivative of Step (a) toform an O-protected derivative of the structural formula

[0037] (c) condensing the O-protected derivative of Step (b) with anamine of the structural formula

[0038] to produce an O-protected derivative of the structural formula

[0039] (d) deprotecting the O-protected derivative of Step (c) to formthe compound of the structural formula

[0040] wherein:

[0041] HET is a heterocyclic moiety selected from the group consistingof oxazolyl, pyrazolyl, and thiazolyl; and

[0042] P is an O-protecting moiety selected from the group consisting of—SiR¹R²R³, —CH₂Ph, —CH₂(p—CH₃OPh), —CH(OCH₂CH₃)CH₃, and

[0043] wherein R¹, R², and R³ are, independently, (C₁-C₆)alkyl, orphenyl.

[0044] Preferably, P is —SiR¹R² R³, and HET is a heterocyclic moietyselected from the group consisting of 2-oxazolyl, 4-oxazolyl,3-pyrazolyl, 4-pyrazolyl, 2-thiazolyl, and 4-thiazolyl. The processwherein P represents —SiR¹R²R³, wherein R¹ and R² are both —CH₃, and R³is —C(CH₃)₃ is especially preferred.

[0045] The stereospecific reduction step, denoted hereinabove as Step(a), preferably employs a fungal reducing agent. Generally, the use offungal and/or microbial reducing agents in the stereospecificbiotransformation of pharmaceutical intermediates is known. See, forexample, R. N. Patel, Advances in Applied Microbiology, 43, 91-140(1997). Specifically, the stereospecific reduction of α-haloketones withvarious microorganisms is also generally known. See, for example, R. N.Patel, et al., JAOCS, 75 (11),1473-1482 (1998), which discloses the useof Agrobacterium tumefaciens ATCC 15955, Alcaligenes eutrophus ATCC17697, Arthrobacter petroleophagus ATCC 21494, Debaryomyces hanseniiATCC 66354, Mycobacterium sp. ATCC 29676, Rhodococcus rhodochorous ATCC14347, Hansenula anomala SC 13833, H. anomala ATCC 16142, H. saturnus SC13829, and Spingomonas paucimobilis SC 16113 in the stereospecificreduction of α-bromoketones. The fungal reducing agent utilized inreduction Step (a) of the instant invention preferably comprises Absidiacylindrospora ATCC 22751 (American Type Culture Collection, Rockville,Md.). The aforementioned reduction step affords the corresponding(R)-bromoalcohol in a highly enantioselective yield, i.e. >90%enantiomeric excess. Preferably, the (R)-bromoalcohol so formed in thestereospecific reduction Step (a) is then isolated, either as a freebase, or an acid addition salt thereof.

[0046] The (R)-bromoalcohol product formed in the stereospecificreduction Step (a) is then O-protected. Synthetic methods of protectingalcohol functional groups are well-known to one of ordinary skill in theart and may comprise, for example, functionalizing the alcohol as asilyl, ether, or ester derivative thereof. Although any conventionalO-protecting group that is compatible with the reaction conditionsemployed in subsequent synthetic steps may be employed in the processesof the present invention, the (R)-bromoalcohol product of Step (a) ispreferably protected as an O-silyl ether derivative. The preferredO-silylation step, generically denoted hereinabove as Step (b), may beeffected according to standard methodologies that will be known to oneof ordinary skill in the art. Such preferred O-silylation is typicallyeffected by treatment of the (R)-bromoalcohol with an appropriatelysubstituted silylating agent. Such silylating agents may comprise, forexample, those silyl derivatives of the formula R¹R²R³Si—X, wherein Xcomprises an appropriate leaving group. Preferably, the silylating agentcomprises a reactant of the formula R¹R² R³Si—X, wherein X is a leavinggroup selected from the group consisting of halogen (e.g., chloro orbromo), cyano, imidazolyl, triflate (trifluoromethanesulfonate), and thelike. However, other silylating agents, that may be employed inaccordance with the processes of the instant invention, will also beknown to one of ordinary skill in the art. Preferably R¹, R², and R³,within the definition of the protected alcohol moiety —OSiR¹R²R³ are,independently, (C₁-C₆)alkyl, or phenyl. The O-silyl ether derivativewherein R¹ and R² are both —CH₃, and R³ is —C(CH₃)₃ is especiallypreferred.

[0047] Typically, such O-silylation is effected by condensing thealcohol to be protected with the silylating agent in the presence of asuitable organic base, for example, an alkylamine, such astriethylamine, N,N-diisopropylethylamine (Hunig's base), or aheterocyclic amine, such as imidazole or diazabicyclo[5.4.0]undec-7-ene(DBU), in a halogenated hydrocarbon solvent, such as dichloromethane.Alternatively, a polar, aprotic solvent, such as dimethylformamide ordimethylsulfoxide may also be employed. With respect to the O-silylationreaction of the present invention, dimethylformamide is preferred.Typically, such silylation is effected by stirring the reactants at, orabout, room temperature for an extended period of time, i.e. overnight.However, such silylation may also be performed at greater, or lesser,than ambient temperature, where appropriate.

[0048] For a detailed discussion of methods of protecting alcoholfunctional groups, including those preferred methods employingsilylating agents see, for example, T. W. Greene, et al., ProtectiveGroups in Organic Synthesis, John Wiley & Sons, New York, N.Y. (1991),and the references cited therein.

[0049] The O-protected derivative so formed in Step (b) is thencondensed in Step (c) with an amine of the structural formula

[0050] to provide a product of the structural formula

[0051] The aforementioned condensation Step (c) may be carried out understandard reaction conditions known to one of ordinary skill in the art.Preferably, the protected (R)-bromoalcohol and the amine are condensedin the presence of a suitable organic base, for example, an alkylamine,such as triethylamine, N,N-diisopropylethylamine (Hunig's base), in apolar, aprotic solvent, such as dimethylsulfoxide. Such condensation istypically effected at an elevated temperature, preferably in the generalrange of from about 40° to about 120° C. Preferably R¹, R², and R³,within the definition of the preferred moiety —SiR¹R²R³ are,independently, (C₁-C₆)alkyl, or phenyl. The process where R¹ and R² areboth —CH₃, and R³ is —C(CH₃)₃ is especially preferred.

[0052] The amine compound utilized in Step (c) hereinabove may beprepared according to an exemplary process for preparing a compound ofthe structural formula

[0053] or an acid addition salt thereof, which process comprises thesteps of:

[0054] (a′) functionalizing a compound of the structural formula

[0055] to provide a compound of the structural formula

[0056] (b′) defunctionalizing the compound so formed in Step (a′) toprovide the compound of the structural formula

[0057] wherein:

[0058] HET is a heterocyclic moiety selected from the group consistingof oxazolyl, pyrazolyl, and thiazolyl. Preferably, HET represents aheterocyclic moiety selected from the group consisting of 2-oxazolyl,4-oxazolyl, 3-pyrazolyl, 4-pyrazolyl, 2-thiazolyl, and 4-thiazolyl.

[0059] In the functionalization step, denoted as Step (a′) hereinabove,a phenolic compound of the structural formula

[0060] is functionalized to provide a carbamate of the structuralformula

[0061] Such phenolic compounds, which may be prepared according toliterature methods or, alternatively, according to the syntheticprocedures disclosed hereinbelow, are most conveniently functionalizedin Step (a′) by the reaction thereof with a compound having the generalformula PhCH₂OCONHCH₂CH₂—Y, wherein Y comprises an appropriate leavinggroup. Exemplary leaving groups comprise those selected from the groupconsisting of tosylate (p-toluenesulfonate), mesylate(methanesulfonate), halogen (e.g., bromo, chloro, or iodo), and thelike. A mesylate leaving group is generally preferred. The compound ofthe general formula PhCH₂OCONHCH₂CH₂—Y, wherein Y is mesylate may beprepared as disclosed in C. A. Townsend, et al., Tetrahedron, 47, 2591(1991). Functionalization of the phenolic compound is preferablyeffected in a polar, aprotic solvent, such as dimethylsulfoxide, in thepresence of an inorganic base, for example, potassium carbonate. Thefunctionalization is typically effected at an elevated temperature,generally in the general range of from about 40° to about 120° C.

[0062] The carbamate derivative so formed in functionalization Step (a′)hereinabove is then defunctionalized in Step (b′) to provide a compoundof the structural formula

[0063] Such defunctionalization of the carbamate product formed in Step(a′) may be carried out according to established methods. For example,the carbamate may be defunctionalized by catalytic hydrogenationemploying a suitable metallic catalyst, such as a nickel salt, or acomplex thereof, a palladium salt, or a complex thereof, or platinum, ora complex thereof. Preferably, the defunctionalization is effected in apolar, protic solvent, such as methanol, using ammonium formate andformic acid in the presence of a metallic catalyst, preferably,palladium on activated carbon. Such defunctionalization is normallyperformed at an elevated temperature, preferably at the refluxtemperature of the solvent employed.

[0064] The amine product thus formed in Step (b′) is then preferablyisolated, either in the form of the free base, or in the form of an acidaddition salt thereof. Conventional techniques of isolating such freebase will be known to one of ordinary skill in the art. Likewise, theacid addition salt of the amine product may also be prepared accordingto known methods, for example, by treatment of the isolated free basewith a conjugate organic acid, such as succinic, tartaric, acetic,citric, maleic, methanesulfonic, or p-toluenesulfonic acid, and thelike, or a conjugate inorganic acid, such as hydrochloric, hydrobromic,sulfuric, or nitric acid, and the like. As was previously disclosedhereinabove, facile product isolation and augmented purity are normallysatisfactorily achieved where such salt formation is carried out in areaction-inert solvent, such as a non-solvent from which the desiredsalt precipitates upon formation, or in a solvent from which the formedsalt precipitates upon subsequent addition of a non-solvent.

[0065] The deprotection step, denoted hereinabove as Step (d), may beperformed according to standard methods that will be known to one ofordinary skill in the art. The preferred —O—SiR¹R²R³ derivative formedin Step (c) is preferably deprotected by the reaction thereof with asuitable alkylammonium fluoride, such as tetrabutylammonium fluoride.Such deprotection may be effected at ambient temperature in an aproticsolvent, for example, tetrahydrofuran. For a detailed discussion ofmethods of deprotecting O-silyl ethers see, for example, T. W. Greene,et al., supra, and the references cited therein.

[0066] The deprotected product of Step (d) is then preferably isolated,either in the form of the free base or, if desired, in the form of apharmaceutically acceptable salt, or a hydrate of such pharmaceuticallyacceptable salt. Such isolation may be effected according towell-established methods. Likewise, the pharmaceutically acceptable saltmay also be prepared according to known methods including, for example,treatment of the isolated free base with a conjugate organic acid, suchas succinic, tartaric, acetic, citric, maleic, methanesulfonic, orp-toluenesulfonic acid, and the like. Alternatively, a conjugateinorganic acid, such as hydrochloric, hydrobromic, sulfuric, or nitricacid, and the like, may also be employed. The tosylate, i.e.,p-toluenesulfonate, salt, abbreviated in the instant description andappendant claims as TsOH, of the deprotected product formed in Step (d)is especially preferred. For purposes of facilitating product isolationand augmenting purity, such salt formation is preferably carried out ina reaction-inert solvent, for example, a non-solvent from which thedesired salt precipitates upon formation or, more preferably, in asolvent from which the formed salt precipitates upon subsequent additionof a non-solvent.

[0067] One of ordinary skill in the art will further appreciate thatpharmaceutically acceptable salts may form hydrated forms thereof, andsuch hydrated forms are embraced within the scope of the presentinvention. Hydrates of pharmaceutically acceptable salts may be preparedaccording to well-known methods including, for example, sublimation,crystallization of the hydrate from a single solvent, formation of thehydrate by evaporation from a binary mixture, vapor diffusion, thermaltreatment, and the like. For a detailed discussion of methods ofpreparing hydrates of pharmaceutically acceptable salts see, forexample, J. Keith Guillory, Polymorphism in Pharmaceutical Solids,Chapter 5, “Generation of Polymorphs, Hydrates, Solvates, and AmorphousSolids”, pp. 183-219, Marcel Dekker, Inc. (1999). In the practice of thecrystal forms, pharmaceutical compositions, and methods of the instantinvention, the monohydrate of the tosylate salt of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol ispreferred.

[0068] In one aspect, the invention provides an anhydrous crystal formof the tosylate salt of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol,which tosylate salt is represented by the structural formula

[0069] In another aspect, the invention provides a crystal form of themonohydrate of the tosylate salt of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol,which monohydrate is represented by the structural formula

[0070] In another aspect, the invention provides a process for preparingan anhydrous crystal form of the tosylate salt of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol,which process comprises the steps of:

[0071] (a) preparing a solution of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol in areaction-inert solvent; and

[0072] (b) treating the solution so formed with p-toluenesulfonic acidmonohydrate such that a final stoichiometric ratio of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol top-toluenesulfonic acid monohydrate of about 1:1 is obtained.

[0073] A solution of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol isprepared by dissolution thereof in a reaction-inert solvent. Suchreaction-inert solvent is preferably a C₁-C₆ alkanol, for example,methanol, ethanol, or isopropanol. Methanol is generally preferred. Thesolution of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol thusformed is then treated with p-toluenesulfonic acid monohydrate in amanner such that a final stoichiometric ratio of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol top-toluenesulfonic acid monohydrate of about 1:1 is obtained. Uponobtention of the above desired stoichiometric ratio, the solution isstirred, preferably at, or about, room temperature, for an additionalperiod of from about 30 minutes to about 3 hours, whereuponprecipitation of the anhydrous tosylate salt is essentially complete.Seeding of the reaction solution may be desirable and/or appropriate ininstances where precipitation of the crystalline anhydrous tosylate salthas not occurred within a reasonable time after complete addition ofp-toluenesulfonic acid monohydrate. Furthermore, addition of anon-solvent, such as diethyl ether, to the reaction mixture may also bedesirable and/or appropriate where precipitation of the anhydroustosylate salt is incomplete. The techniques of reaction seeding andaddition of non-solvents will be readily recognized by one of ordinaryskill in the art as valuable, conventional methods of isolatingpharmaceutically acceptable salts.

[0074] The crystal form of the anhydrous tosylate salt so prepared isthen preferably isolated, preferably by filtration, according tostandard techniques that will be well known to one of ordinary skill inthe art.

[0075] In another aspect, the invention provides a process for preparinga crystal form of the monohydrate of the tosylate salt of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol,which process comprises contacting(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt with water, optionally in the presence of areaction-inert solvent.

[0076] The crystal form of the monohydrate of the tosylate salt of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol maybe prepared by exposing the tosylate salt to water vapor or,alternatively, by admixing a suspension, a solution, or a partialsolution of the tosylate salt in water, optionally in the presence of areaction-inert solvent. Accordingly, a volume ratio of water to theoptional inert solvent may range generally from about 100:0 to about1:10. Such monohydrate formation is typically effected at a temperaturerange of from about 10° C. to about 60° C., preferably at, or about,room temperature, for a period of from about 30 minutes to about 14days. Where such monohydrate formation is effected by exposing thetosylate salt to water vapor, relative humidities typically employed arein the general range of from about 59% to about 100%, preferably fromabout 75% to about 100%. Preferably, monohydrate formation is effectedby admixing a partial solution of the tosylate salt in water in thepresence of a reaction-inert solvent at about room temperature for aperiod of from about 30 minutes to about 12 hours.

[0077] Where the preferred hydrate formation is effected in the presenceof a reaction-inert solvent, the volume ratio of water to inert solventmay range generally from about 0.4:10 to about 7.5:10. A preferredvolume ratio of water to inert solvent is about 1:10. The optionalreaction-inert solvent employed in the monohydrate formationhereinabove, is preferably selected from the group consisting of a C₁-C₆alkanol; for example, methanol, ethanol, or isopropanol; ethyl acetate;acetone; acetonitrile; or tetrahydrofuran. The preferred solvent istetrahydrofuran. The crystal form of the monohydrate of the tosylatesalt so prepared is then preferably isolated, preferably by filtration,according to standard techniques that will be well known to one ofordinary skill in the art.

[0078] In yet another aspect, the instant invention further providespharmaceutical compositions comprising the crystal forms of the instantinvention, and a pharmaceutically acceptable carrier, vehicle, ordiluent.

[0079] In yet another aspect, the invention further provides methods oftreating β₃-adrenergic receptor-mediated diseases, conditions, ordisorders in a mammal which comprise administering to a mammal in needof such treatment a therapeutically effective amount of the crystalforms of the instant invention, or a pharmaceutical compositioncomprising such crystal forms. Preferably, the β₃-adrenergicreceptor-mediated disease, condition, or disorder is selected from thegroup consisting of obesity, diabetes, irritable bowel syndrome,inflammatory bowel disease, esophagitis, duodenitis, Crohn's Disease,proctitis, asthma, intestinal motility disorder, ulcer, gastritis,hypercholesterolemia, cardiovascular disease, urinary incontinence,depression, prostate disease, dyslipidemia, and airway inflammatorydisorder. The methods wherein the β₃-adrenergic receptor-mediateddiseases, conditions, or disorders are selected from the groupconsisting of obesity, diabetes, urinary incontinence, and irritablebowel syndrome are especially preferred.

[0080] The crystal forms of the instant invention, and thepharmaceutical compositions comprising such crystal forms, furtherpossess utility for increasing lean meat content in edible animals,i.e., ungulate animals such as cattle, swine, and the like, as well aspoultry. Accordingly, the invention also provides methods of increasinglean meat content in an edible animal which methods compriseadministering to the edible animal a lean meat increasing amount of thecrystal forms of the instant invention, or a pharmaceutical compositioncomprising such crystal forms.

[0081] The crystal forms of the instant invention can be administered toa patient at dosage levels in the range of from about 0.01 to about1,000 mg per day. For a normal adult human having a body mass of about70 kg, a dosage in the range of from about 0.01 to about 300 mg istypically sufficient. However, some variability in the general dosagerange may be required depending upon the age and weight of the subjectbeing treated, the intended route of administration, and the like. Thedetermination of dosage ranges and optimal dosages for a particularpatient is well within the ability of one of ordinary skill in the arthaving benefit of the instant disclosure.

[0082] According to the methods of the invention, the crystal forms ofthe instant invention are administered to a mammal in need of treatmenttherewith, preferably in the form of a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier, vehicle, or diluent.Accordingly, such crystal forms can be administered to a mammal in anyconventional oral, rectal, transdermal, parenteral, (e.g., intravenous,intramuscular, or subcutaneous), intracisternal, intravaginal,intraperitoneal, intravesical, local (e.g., powder, ointment, or drop),buccal, or nasal dosage form.

[0083] Compositions suitable for parenteral injection may comprisepharmaceutically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions, or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents,solvents, or vehicles include water, ethanol, polyols (e.g., propyleneglycol, polyethylene glycol, glycerol, and the like), suitable mixturesthereof, vegetable oils e.g., olive oil), and injectable organic esterssuch as ethyl oleate. Proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

[0084] These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and dispersing agents. Prevention of microorganismcontamination of these compositions can be effected with variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. It may also bedesirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of injectablepharmaceutical compositions can be effected by the use of agents capableof delaying absorption, for example, aluminum monostearate, and gelatin.

[0085] Solid dosage forms for oral administration include capsules,tablets, powders, and granules. In such dosage forms, the crystal formsof the instant invention are admixed with at least one inert customarypharmaceutical excipient (or carrier) such as sodium citrate, ordicalcium phosphate, or (a) fillers or extenders; (b) binders, as forexample, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, as forexample, glycerol; (d) disintegrating agents, as for example, agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certaincomplex silicates, and sodium carbonate; (e) solution retarders, as forexample, paraffin; (f) absorption accelerators, as for example, cetylalcohol and glycerol monostearate; (h) adsorbents, as for example,kaolin and bentonite; and/or (i) lubricants, as for example, talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate, or mixtures thereof. In the case of capsules andtablets, the dosage forms may also comprise buffering agents.

[0086] Solid compositions of a similar type may also be employed asfillers in soft or hard filled gelatin capsules using such excipients aslactose or milk sugar, as well as high molecular weight polyethyleneglycols, and the like.

[0087] Solid dosage forms such as tablets, dragees, capsules, andgranules can be prepared with coatings and shells, such as entericcoatings and others well known in the art. They may also contain certainopacifying agents, and can be of such composition that they release theactive compound or compounds in a delayed manner. Examples of embeddingcompositions that can also be employed are polymeric substances andwaxes. The crystal forms of the instant invention can also beincorporated in micro-encapsulated form, if appropriate, with one ormore of the above-mentioned excipients.

[0088] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, solutions, suspensions, syrups,and elixirs. In addition to the tosylate salt, or the monohydrate ofsuch salt, the liquid dosage form may contain inert diluents commonlyused in the art, such as water or other solvents, solubilizing agentsand emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oil, in particular,cottonseed oil, groundnut oil, corn germ oil, castor oil, and sesameseed oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols,and fatty acid esters of sorbitan, or mixtures of these substances, andthe like.

[0089] Besides such inert diluents, the compositions may also compriseadjuvants, such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and perfuming agents.

[0090] Suspensions of the crystal forms of the instant invention mayfurther comprise suspending agents, as for example, ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite,agar-agar, and tragacanth, or mixtures of these substances, and thelike.

[0091] Compositions for rectal or vaginal administration preferablycomprise suppositories, which can be prepared by admixing the crystalforms of the instant invention with suitable non-irritating excipientsor carriers such as cocoa butter, polyethylene glycol, or a suppositorywax, which are solid at normal room temperature, but liquid at bodytemperature and, therefore, melt in the rectum or vaginal cavity therebyreleasing such crystal forms.

[0092] Dosage forms for topical administration may comprise ointments,powders, sprays, and inhalants. The crystal forms of the instantinvention are admixed under sterile conditions with a pharmaceuticallyacceptable carrier, and any preservatives, buffers, or propellants thatmay also be required. Opthalmic formulations, eye ointments, powders,and solutions are also intended to be included within the scope of thepresent invention.

[0093] The following paragraphs describe exemplary formulations,dosages, etc. useful for non-human animals. In such animals,administration of the crystal forms of the instant invention can beeffected orally, or non-orally, for example, by injection.

[0094] An amount of the crystal forms of the instant invention isadministered such that an effective dose is received, generally a dailydose which, when administered orally to an animal, is usually betweenabout 0.01 to about 1,000 mg per kg body mass, preferably between about0.01 to about 300 mg per kg body mass.

[0095] Conveniently, the crystal forms can be carried in the drinkingwater such that a therapeutic dosage of such forms are ingested with thedaily water supply. The crystal forms can be metered directly intodrinking water, preferably in the form of a liquid, water-solubleconcentrate, such as an aqueous solution of such crystal forms.

[0096] Conveniently, the crystal forms of the instant invention can alsobe added directly to the feed, as such, or in the form of an animal feedsupplement, also referred to as a premix or concentrate. A premix orconcentrate in a carrier is more commonly employed for the inclusion ofthe crystal forms of the instant invention in the feed. Suitablecarriers are liquid or solid, as desired, such as water, various mealssuch as alfalfa meal, soybean meal, cottonseed oil meal, linseed oilmeal, corncob meal, molasses, urea, bone meal, and mineral mixes such asare employed commonly in poultry feeds. A particularly effective carrieris the respective animal feed itself; that is a small portion of suchfeed. The carrier facilitates uniform distribution of the crystal formsof the instant invention in the finished feed with which the premix isblended. It is important that such salt, or such hydrate, be thoroughlyblended into the premix and, subsequently, the feed. In this respect,the crystal forms may be dispersed or dissolved in a suitable oilyvehicle such as soybean oil, corn oil, cottonseed oil, and the like, orin a volatile organic solvent and then blended with the carrier. It willbe appreciated that the proportions of such crystal forms in theconcentrate are capable of wide variation since the amount(s) thereof inthe finished feed may be adjusted by blending the appropriate proportionof premix with the feed to obtain a desired level of such crystal forms.

[0097] High potency concentrates may be blended by the feed manufacturerwith a proteinaceous carrier such as soybean oil meal and other meals,as described hereinabove, to produce concentrated supplements, which aresuitable for direct feeding to animals. In such instances, the animalsare permitted to consume the usual diet. Alternatively, suchconcentrated supplements may be added directly to the feed to produce anutritionally balanced, finished feed containing a therapeuticallyeffective amount or level of the crystal forms of the instant invention.The mixtures are thoroughly blended by standard procedures, such as in atwin shell blender, to ensure homogeniety.

[0098] If the supplement is used as a top dressing feed, it likewisehelps to ensure uniformity of distribution of the crystal forms acrossthe top of the dressed feed.

[0099] Drinking water and feed effective for increasing lean meatdeposition and for increasing lean meat to fat ratio are generallyprepared by mixing the crystal forms of the instant invention with asufficient amount of animal feed to provide from about 10⁻³ to about 500ppm of such salt, or such monohydrate, in the feed or water.

[0100] The preferred medicated swine, cattle, sheep, and goat feedgenerally contain from about 1 to about 400 grams of the crystal formsof the instant invention per ton of feed, the optimum amount for theseanimals usually being about 50 to about 300 grams per ton of feed.

[0101] The preferred poultry and domestic pet feeds usually containabout 1 to about 400 grams and, preferably, about 10 to about 400 gramsof the crystal forms per ton of feed.

[0102] For parenteral administration in animals, the crystal forms ofthe instant invention may be prepared in the form of a paste or a pelletand administered as an implant, usually under the skin of the head orear of the animal in which increase in lean meat deposition andimprovement in lean meat to fat ratio is sought.

[0103] In general, parenteral administration involves injection of asufficient amount of the crystal forms of the instant invention toprovide the animal with about 0.01 to about 20 mg per kg body mass perday. The preferred dosage for poultry, swine, cattle, sheep, goats, anddomestic pets is in the range of from about 0.05 to about 10 mg per kgbody mass per day.

[0104] Paste formulations can be prepared by dispersing the crystalforms of the instant invention in a pharmaceutically acceptable oil,such as peanut oil, sesame seed oil, and the like.

[0105] Pellets containing an effective amount of the crystal forms ofthe instant invention can be prepared by admixing such forms with adiluent such as carbowax, carnuba wax, and the like, and a lubricant,such as magnesium or calcium stearate, can be added to improve thepelleting process.

[0106] It will be appreciated that more than one pellet may beadministered to an animal to achieve the desired dosage level which willprovide the increase in lean meat deposition and improvement in leanmeat to fat ratio desired. Moreover, it has been determined thatimplants may also be made periodically during the animal treatmentperiod in order to maintain the proper drug level in the animal's body.

[0107] The present invention has several advantageous veterinaryfeatures. For the pet owner or veterinarian who wishes to increaseleanness and/or trim unwanted fat from pet animals, the instantinvention provides methods by which this may be accomplished. Forpoultry and swine breeders, utilization of the methods of the presentinvention yields leaner edible animals which command higher sale pricesfrom the meat industry.

EXAMPLES

[0108] The present invention is illustrated by the following Examples.It is to be understood, however, that the invention is not limited tothe specific details of these examples, as other variations thereof willbe known, or apparent in light of the instant disclosure, to one ofordinary skill in the art.

Preparation of Intermediates Preparation of Intermediate(R)-2-Bromo-1-pyridin-3-yl-ethanol (I-1):

[0109]

[0110] 2-Bromo-1-pyridin-3-yl-ethanone hydrobromide (G. B. Davies, etal., Aust. J. Chem., 42, 1735 (1989)) was contacted with cultures ofAbsidia cylindrospora ATCC 22751 grown in Fernbach flasks, or fermentorcultures containing medium A (40 g/l corn steep solids and 20 g/lglucose adjusted to pH 4.85 prior to autoclaving). Fernbach flasks (8),each containing 500 ml of medium A, were inoculated with 5 ml of a seedculture of Absidia cylindrospora ATCC 22751. The seed cultures of A.cylindrospora were prepared in two 300 ml conical flasks, eachcontaining 40 ml of medium A. These seed cultures were inoculated with aspore stock of A. cylindrospora and agitated (210 rpm) for about 24hours at 29° C. After agitation for a total of about 41 hours at about29° C., 25 ml of a 20 g/l aqueous solution of the hydrobromide salt of2-bromo-1-pyridin-3-yl-ethanone was added to each of the Fernbach flaskcultures. The flasks were agitated for about an additional 5 hours afterwhich the contents of the flasks were combined and centrifuged to removesolid materials.

[0111] Two cultures of Absidia cylindrospora ATCC 22751 were grown infermentors containing 8 l of medium A. The fermentors were eachinoculated with a single culture of A. cylindrospora grown in Fernbachflasks containing 400 ml of medium A. The Fernbach flask cultures wereinoculated with 1.8 ml of spore stock of A. cylindrospora ATCC 22751 andagitated (200 rpm) for about 40 hours at about 29° C. After about 24hours, the two fermentor cultures were treated with an aqueous solutionof 2-bromo-1-pyridin-3-yl-ethanone hydrobromide (30 g/l) which resultedin the addition of 8 g of 2-bromo-1-pyridin-3-yl-ethanone hydrobromideto one fermentor, and 16 g of 2-bromo-1-pyridin-3-yl-ethanonehydrobromide to the other fermentor. The fermentor culture that received8 g of 2-bromo-1 -pyridin-3-yl-ethanone hydrobromide was harvested about24 hours following substrate addition, while the other fermentor washarvested about 5 hours following substrate addition. The contents ofboth fermentor cultures were centrifuged to remove solid materials.

[0112] The supernatant phases from the eight Fernbach flask cultures andthe two fermentor cultures were combined, filtered through filter paper,and passed through a column containing 737 g of XAD-16® resin (Rohm &Haas; Philadelphia, Pa.). The resin was then eluted with mixtures ofmethanol and water (1110% methanol, 1120% methanol, 1 l 30% methanol, 1l 50% methanol, 3×1 l 80% methanol, and 1 l 100% methanol) and fractionswere collected. These fractions were analyzed by HPLC on a 4.6×150 mmKromasil® C4 column (Phenomenex; Torrance, Calif.), eluting with 10 mMammonium acetate:acetonitrile (76.5:23.5, v/v) at 1.0 ml/minute, andthose fractions found to contain desired product (10% methanol −80%methanol) were pooled, concentrated to remove solvent, and extractedwith ethyl acetate. The ethyl acetate extracts were combined,concentrated to about 600 ml, dried with magnesium sulfate, andfiltered. This material was divided into several portions and thenpurified by flash chromatography on silica gel cartridges (1.2×7.5 cmand 4×15 cm, Biotage; Charlottesville, Va.) eluting with ethyl acetateand hexane mixtures containing 0.1% acetic acid(ethylacetate:hexane:acetic acid; 60:40:0.1; v/v/v). Fractions containingdesired product were concentrated to give 1.93 g (9.6%) of titlecompound as a light yellow oil, α_(D)=−16.4° (c=0.53, methanol). ChiralHPLC analysis of the product on 4.6×250 mm Chiralcel® OD column (ChiralTechnologies; Exton, Pa.) eluting with hexanes:isopropyl alcohol (9:1,v/v) at 1.5 ml/minute revealed an enantiomeric excess of 91.2%.

[0113]¹HNMR (400 mHz, d₆-DMSO): δ8.55 (d, 1H, J=2.1 Hz), 8.44 (dd, 1H,J=1.7, 4.6 Hz), 7.75 (dd, 1H, J=2.5, 4.2 Hz), 7.33 (m, 1H), 5.93 (d, 1H,J=4.6 Hz), 4.85 (m, 1H), 3.60 (ddd, 2H, J=5.0, 10.4, 14.9 Hz). GC-MS(m/z, %): 201/203 (M⁺, 10), 108 (100).

Preparation of Intermediate(R)-3-(2-Bromo-1-(tert-butyl-dimethyl-silanyl)-ethyl)-pyridine (I-2):

[0114]

[0115] To a stirred solution of 1.54 g (7.61 mmol) of(R)-2-bromo-1-pyridin-3-yl-ethanol I-1 in 20 ml of dryN,N-dimethylformamide at room temperature was added 1.55 g (22.83 mmol)of imidazole followed by 1.72 g (11.4 mmol) of tert-butyldimethylsilylchloride. The mixture was stirred at room temperature for about 18 hoursand then an additional 1.55 g (22.83 mmol) of imidazole and 1.72 g (11.4mmol) of tert-butyldimethylsilyl chloride were added, and the mixturewas stirred at room temperature for about an additional 24 hours. Themixture was poured into 200 ml of water and extracted with ethyl acetate(2×200 ml). The organic extracts were combined, washed successively withwater (1×40 ml), brine (1×40 ml), then dried over magnesium sulfate andconcentrated in vacuo to furnish an oil. Chromatography on silica geleluting with ethyl acetate:hexanes (2:3, v/v) provided 1.41 g (58%yield) of the desired title compound as a clear oil, α_(D)=−51.5(c=0.60, chloroform). Chiral HPLC analysis of the product on 4.6×250 mmChiralcel® OD column (Chiral Technologies; Exton, Pa.) eluting withhexanes:isopropyl alcohol (7:3, v/v) at 1.0 ml/minute revealed anenantiomeric excess of 91.3%.

[0116]¹HNMR (400 mHz, CDCl₃): δ8.58 (s, 1H), 8.55 (m, 1H), 7.70 (d, 1H),7.30 (m, 1H), 4.90 (m, 1H), 3.46 (ddd, 2H, J=1,2, 7.1, 8.3 Hz), 0.87 (s,9H), 0.11 (s, 3H). MS (m/z, %): 316/318 (M⁺, 100).

Preparation of Intermediate [2-(4-Oxazol-4-yl-phenoxy)-ethyl]-carbamicAcid Benzyl Ester (I-3):

[0117]

[0118] A stirred mixture of 290.0 g (1.80 mol) of 4-oxazol-4-yl-phenol(H. Jones, et al., J. Med. Chem., 21, 1110 (1978)), 737.7 g (2.70 mol)of methanesulfonic acid 2-benzyloxycarbonylamino-ethyl ester, and 746.0g (5.40 mol) of potassium carbonate in 4.6 l of dry dimethylsulfoxidewas heated to about 85° C. An additional 500 ml of dimethylsulfoxide wasadded and the viscous slurry was stirred at about 80° C. for about anadditional two hours. The resulting mixture was cooled to about 50° C.,poured into about 1 l of stirred ice water, slurried for about one hour,and then filtered. The wet filter cake was washed with water (2×1 l),and then partially dried by aspiration under vacuum for about two hours.The moist solid was charged into a round-bottomed flask, 6 liters ofmethanol was added, and the mixture was then warmed to about 60° C.where 3 liters of water was added. The heating source was removed, themixture was stirred for about eighteen hours, and then filtered. Thefilter cake was washed with 2:1 methanol/water (v/v; 2×500 ml), and thendried under vacuum at about 40° C. for about eighteen hours. The titlecompound (389.5 g, 64% yield) was obtained as a beige powder.

Preparation of Intermediate 2-(4-Oxazol-4-yl-phenoxy)-ethylamine (I-4):

[0119]

[0120] A stirred mixture of 234.0 g (0.692 mol) of[2-(4-oxazol-4-yl-phenoxy)-ethyl]-carbamic acid benzyl ester I-3, 295.1ml (3.097 mol) of 1,4-cyclohexadiene, and 93.60 g of 10% Pd/C (50% waterwet) in 5.6 l of methanol was stirred at room temperature for abouttwenty-two hours. The mixture was filtered through a pad of diatomaceousearth (13×3 cm), and the filter cake was then washed with 12 l of 100:1v/v methanol/triethylamine. The filtrate was evaporated in vacuo, and tothe residual solid was added 250 ml of toluene. The mixture was stirredat room temperature for about thirty minutes, 2.5 l of hexanes was thenadded over a period of about five to ten minutes, and the resultingslurry was then stirred for about one hour. The mixture was filtered,and the filter cake was then washed with a mixture of 1:10toluene/hexanes (3×100 ml), and the solid was dried under vacuum atabout 50° C. for about eighteen hours. The title compound (115 g, 81.5 %yield) was obtained as a white powder.

Preparation of(R)-(2-tert-Butyl-dimethylsilanoxy)-2-pyridin-3-yl-ethyl)-(2-(4-oxazol-4-yl-phenoxy)-ethyl)-amine(I-5):

[0121]

[0122] A stirred mixture of 1.24 g (3.91 mmol) of(R)-3-(2-bromo-1-(tert-butyl-dimethyl-silanyl)-ethyl)-pyridine I-2, 1.6g (7.83 mmol) of 2-(4-oxazol-4-yl-phenoxy)-ethylamine I-4, and 1.4 ml(7.83 mmol) of diisopropylethylamine in 20 ml of dry dimethylsulfoxidewas heated at about 90° C. for about 18 hours. The mixture was pouredinto 400 ml of water and extracted with ethyl acetate (2×400 ml). Theorganic extracts were combined, washed successively with water (2×100ml) and brine (1×100 ml), dried over magnesium sulfate, and concentratedin vacuo to furnish an oil. Chromatography on silica gel eluting withmethanol:dichloromethane (1:19, v/v) yielded 963 mg (56% yield) of thetitle compound as an amber-colored oil, α_(D)=−45.7° (c=0.49,chloroform).

[0123]¹HNMR (400 mHz, CDCl₃): δ8.56 (d, 1H, J=2.1 Hz), 8.50 (dd, 1H,J=1.7, 5.0 Hz), 7.90 (d, 1H, J=0.8 Hz), 7.84 (d, 1H, J=0.8 Hz), 7.65 (m,3H), 7.26 (m, 2H), 6.90 (m, 2H), 4.85 (dd, 1 H, J=3.7, 8.3 Hz), 4.07 (m,2H), 3.01 (dd, 2H, J=4.6, 6.2 Hz), 2.88 (dd, 2H, J=8.3, 12.0 Hz), 2.76(dd, 2H, J=3.7, 11.6 Hz), 0.88 (s, 9H), 0.06 (s, 3H). MS (m/z, %): 441(M⁺+1, 100).

Preparation of Intermediate(R)-2-(2-(4-Oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol(I-6):

[0124]

[0125] To a stirred solution of 646 mg (1.47 mmol) of(R)-(2-tert-butyl-dimethylsilanoxy)-2-pyridin-3-yl-ethyl)-(2-(4-oxazol-4-yl-phenoxy)-ethyl)-amineI-5 in 5 ml of dry tetrahydrofuran at room temperature was added 2.2 ml(2.20 mmol) of 1.0 M tetrabutylammonium fluoride in tetrahydrofuran. Themixture was stirred at room temperature overnight, poured into 100 ml ofwater, and extracted with ethyl acetate (2×100 ml). The organic extractswere combined, washed successively with water (1×20 ml) and brine (1×20ml), dried over magnesium sulfate, and concentrated in vacuo to furnisha solid. Chromatography on silica gel eluting withmethanol:dichloromethane (1:9, v/v) yielded a solid. Trituration with 10ml of ethyl acetate:hexanes (1:1, v/v) afforded 250 mg (52% yield) oftitle compound as a white solid, m.p. 98-100° C., α_(D)=−31.6° (c=0.58,chloroform). Chiral HPLC analysis of the product on 4.6×5 cm ChiralpakAS® column (Chiral Technologies; Exton, Pa.) eluting withacetonitrile:methanol (95:5, v/v) at 1.0 ml/minute revealed anenantiomeric excess of >99.9%.

[0126]¹HNMR (400 mHz, d₆-DMSO): δ8.52 (d, 1H, J=2.1 Hz), 8.47 (d, 1H,J=0.8, 5.0 Hz), 8.41 (dd, 1 H, J=1.7, 4.6 Hz), 8.38 (d, 1 H, J=0.8 Hz),7.70 (m, 3H), 7.30 (m, 1 H), 6.96 (ddd, 2H, J=2.5, 4.6, 9.5 Hz), 5.47(d, 1H, J=3.7 Hz), 4.67 (d, 1H), 4.02 (m, 2H), 2.89 (t, 2H, J=5.4 Hz),2.72 (d, 2H, J=6.2 Hz). MS (m/z, %): 326 (M⁺+1, 100).

[0127] Anal. Calc'd. for C₁₈H₁₉N₃O₃: C, 66.45; H, 5.89; N, 12.91. Found:C, 66.22; H, 5.92; N, 12.83.

Example 1 Preparation of(R)-2-(2-(4-Oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate Salt

[0128]

[0129] A suspension of 228.10 g (0.701 mol) of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol I-6in 1.6 l of methanol was stirred until a complete solution had beenachieved (˜10 minutes), then p-toluenesulfonic acid monohydrate (133.40g, 0.701 mol) was slowly added, and the resulting solution was stirredat room temperature for about thirty minutes. To the resulting slurrywas added 2.4 1 of diethyl ether over a period of about five to tenminutes, and the viscous slurry was stirred vigorously for about anadditional one hour. The mixture was filtered, and the filter cake wasthen washed with a mixture of 2:1 diethyl ether-methanol (v/v, 3×150ml). The product was dried under vacuum at about 40° C. for abouteighteen hours to afford 303.55 g (87% yield) of the title anhydroustosylate salt in the form of off-white flakes, m.p. 159.7 - 161.5° C.

[0130] Anal. Calc'd. for C₂₅H₂₇N₃SO₆: C, 60.35; H, 5.47; N, 8.45. Found:C, 60.42; H, 5.53; N, 8.27.

Example 2 Preparation of(R)-2-(2-(4-Oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate Salt, Monohydrate

[0131]

[0132] A 1.025 kg sample of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt from Example 1 was slurried in a mixture of 3 lof tetrahydrofuran and 300 ml of water for about eighteen hours. Thesolid was collected by suction filtration, the filter cake was washedwith about 1 l of tetrahydrofuran, and the resulting solid was driedunder continued suction for about thirty minutes. The solid was thenvacuum dried at a temperature of about 30° C. for about seventy-twohours to afford 768.5 g of the title monohydrate salt.

[0133] Anal. Calc'd. for C₂₅H₂₉N₃SO₇: C, 58.47; H, 5.77; N, 8.17; S,6.36. Found: C, 58.18; H, 5.70; N, 8.12; S, 6.26.

[0134] The X-ray diffraction patterns of the crystal form of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt, monohydrate, and the anhydrous crystal form of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt were measured with a Siemens D5000 X-raydiffractometer under the following conditions:

[0135] Anode: Cu

[0136] Wavelength 1: 1.54056

[0137] Wavelength 2: 1.54439

[0138] Rel. Intensity: 0.500

[0139] Range #1

[0140] Coupled: 3.000 to 40.000

[0141] Step Size: 0.040

[0142] Step Time: 1.00

[0143] Smoothing: 0.300

[0144] Threshold: 1.0

[0145] The X-ray diffraction patterns thus measured for the crystal formof (R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt, monohydrate and the crystal form of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt are summarized below in Tables 1 and 2,respectively. TABLE I(R)-2-(2-(4-Oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin- 3-yl-ethanol),p-toluenesulfonate salt, monohydrate Angle d-value Angle d-value Angle(2 Theta) (Å) I (2 Theta) (Å) I (2 Theta) d-value (Å) I 5.7 15.6 100.019.0 4.7 2.8 28.8 3.1 5.7 8.3 10.6 1.6 19.8 4.5 18.1 30.1 3.0 9.3 9.79.1 1.0 20.3 4.4 11.1 30.6 2.9 10.1 11.6 7.6 2.6 20.6 4.3 8.3 31.4 2.85.1 12.0 7.3 5.5 21.4 4.1 16.7 32.4 2.8 2.8 13.1 6.8 4.1 23.1 3.8 25.633.0 2.7 5.2 13.9 6.4 9.7 23.5 3.8 10.3 33.7 2.7 2.2 14.5 6.1 6.3 24.53.6 16.2 34.3 2.6 3.9 15.2 5.8 4.5 25.0 3.6 6.9 35.2 2.5 1.6 16.0 5.59.7 25.7 3.5 10.9 35.9 2.5 2.1 16.5 5.4 17.0 26.4 3.4 4.9 37.1 2.4 2.517.2 5.1 53.6 27.3 3.3 8.6 41.5 2.2 0.8 18.1 4.9 3.7 28.1 3.2 2.0

[0146] TABLE 2 (R)-2-(2-(4-Oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol), p-toluenesulfonate salt Angle d-value Angle d-value Angled-value (2 Theta) (Å) I (2 Theta) (Å) I (2 Theta) (Å) I 6.0 14.7 58.820.4 4.3 3.9 29.4 3.0 0.8 9.0 9.8 1.1 21.2 4.2 9.3 30.1 3.0 4.1 12.1 7.330.3 22.0 4.0 1.7 30.5 2.9 2.1 13.1 6.7 0.9 22.5 3.9 0.8 31.5 2.8 0.613.7 6.4 2.4 23.2 3.8 0.7 33.0 2.7 1.6 15.1 5.8 3.3 24.3 3.7 20.2 33.62.7 10.9 16.2 5.5 0.6 24.8 3.6 3.3 36.8 2.4 7.0 18.1 4.9 100.0 25.5 3.51.2 38.9 2.3 2.2 19.9 4.5 4.3 27.4 3.3 21.8

What is claimed is:
 1. A crystal form of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt.
 2. The crystal form of claim 1 wherein saidcrystal form has high-intensity X-ray diffraction peaks at diffractionangles (2 theta) of about 6.0 and 18.1, and X-ray powder diffractiond-spacings (Å) of about 14.7 and 4.9, respectively.
 3. The crystal formof claim 1 having the following characteristic high-intensitydiffraction peaks at diffraction angles (2 theta) and X-ray powderdiffraction d-spacing (Å): Angle d-value (2 Theta) (æ) I 6.0 14.7 58.812.1 7.3 30.3 18.1 4.9 100 24.3 3.7 20.2 27.4 3.2 21.8


4. The crystal form of claim 1 wherein said crystal form has an onsetmelting temperature of about 160° C.
 5. A crystal form of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt, monohydrate.
 6. The crystal form of claim 5wherein said crystal form has high-intensity X-ray diffraction peaks atdiffraction angles (2 theta) of about 5.7 and 17.2, and X-ray powderdiffraction d-spacings (Å) of about 15.6 and 5.1.
 7. The crystal form ofclaim 5 wherein said crystal form has the following characteristichigh-intensity diffraction peaks at diffraction angles (2 theta), andX-ray powder diffraction d-spacing (Å): Angle d-value (2 Theta) (Å) I5.7 15.6 100 16.5 5.4 17.0 17.2 5.1 53.6 19.8 4.5 18.1 23.1 3.8 25.6


8. A process for preparing crystalline(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt comprising the steps of: (a) preparing asolution of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol in areaction-inert solvent; and (b) treating said solution from step (a)with p-toluenesulfonic acid monohydrate to obtain a final stoichiometricratio of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol top-toluenesulfonic acid monohydrate of about 1:1.
 9. A process forpreparing crystalline(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt, monohydrate comprising the step of contacting(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt with water, optionally in the presence of areaction-inert solvent.
 10. The process of claim 9 wherein saidreaction-inert solvent is tetrahydrofuran.
 11. The process according toclaim 10 wherein said water and said reaction-inert solvent are presentin a ratio of from about 0.4:10 to about 7.5:10.
 12. The process ofclaim 10 wherein said water and said reaction-inert solvent are presentin a ratio of about 1:10.
 13. A pharmaceutical composition comprising(i) a therapeutically effective amount of a crystal form of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt, or a crystal form of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt, monohydrate; and (ii) a pharmaceuticallyacceptable carrier, vehicle, or diluent.
 14. A method of treating aβ₃-adrenergic receptor-mediated disease, condition, or disordercomprising the step of administering to a mammal in need of suchtreatment a therapeutically effective amount of a crystal form of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt; a crystal form of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt, monohydrate; or a pharmaceutical compositioncomprising said a crystal form of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt, or said crystal form of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt, monohydrate.
 15. A method of increasing leanmeat content of an edible animal comprising the step of administering tosaid edible animal a lean meat increasing amount of a crystal form of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt; a crystal form of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt, monohydrate; or a pharmaceutical compositioncomprising said crystal form of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt, or said crystal form of(R)-2-(2-(4-oxazol-4-yl-phenoxy)-ethylamino)-1-pyridin-3-yl-ethanol),p-toluenesulfonate salt, monohydrate.